Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5'exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date there are over 150 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). Presently, there are over 150 pathogenic disease mutations in the POLG gene that cause PEO, ataxia-neuropathy and Alpers syndrome. Mutations in POLG are a major contributor to pediatric and adult mitochondrial diseases. However, the consequences of many POLG mutations are not well understood. We investigated the molecular cause of Alpers syndrome in a patient harboring the POLG mutations A467T in trans with the c.2157+5_+6 gc&#8594;ag substitutions in intron 12. Analysis of transcripts arising from the c.2157+5_+6 gc&#8594;ag allele revealed alternative splicing with an insertion of 30 intronic nucleotides leading to a premature termination codon. These transcripts were subsequently removed through nonsense-mediated decay, leading to haplotype insufficiency due to the prominent expression of the A467T allele and decreased expression of the c.2157+5_+6 gc&#8594;ag allele, which is likely responsible for the Alpers syndrome phenotype. Compared with other POLG-diseases caused by the A467T in trans with either a missense mutation or a null allele, this study reveals that the number of functional POLG alleles is critical to the age of onset. Over seventy different point mutations in POLG cause early onset Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol gamma revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain G848S, T851A, R852C, R853Q, and two Alpers mutations at less conserved positions in the adjacent palm subdomain, Q879H, and T885S. In the first analysis of the pol gamma thumb domain, we biochemically characterized recombinant mutant forms of pol gamma, which revealed that Alpers mutations in the thumb subdomain reduced polymerase activity by more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50 - 70% of wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with pol gammas accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggest a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol gamma and examined the consequences of mitochondrial disease mutations in this region. We developed new methods to facilitate the analysis of yeast microarray data using Cytoscape and constructed an interaction network (interactome) using the curated interaction data available from the Saccharomyces Genome Database and the database of yeast transcription factors. These data were formatted and imported into Cytoscape using semi-automated methods, including Linux-based scripts that simplified the process while minimizing the introduction of processing errors. Using Cytoscape, we illustrated the use of this interactome through the analysis of expression data from a recent yeast diauxic shift experiment. We also reported and briefly described the complex associations among transcription factors that result in the regulation of thousands of genes through coordinated changes in expression of dozens of transcription factors. These cells are thus able to sensitively regulate cellular metabolism in response to changes in genetic or environmental conditions through relatively small changes in the expression of large numbers of genes, affecting the entire yeast metabolome. To further explore the role of POS5, the mitochondrial NADH kinase that we identified in 2003, we examined gene expression in pos5- cells, comparing these data to those from cells containing deletions of superoxide dismutase-encoding genes SOD1 or SOD2. Surprisingly, stress response genes were down-regulated in pos5&#916;, sod1- and sod2- cells, implying that cells infer stress levels from mitochondrial activity rather than sensing reactive oxygen species directly. Additionally, pos5-, but not sod1 or sod2, cells displayed an anaerobic expression profile, indicating a defect in oxygen sensing that is specific to pos5, and is not a general stress response. The pos5- expression profile was found to be quite similar to the hap1- expression profile previously reported, which may indicate a shared mechanism. In 1998 we identified the5'-deoxyribose phosphate (dRP) lyase activity in the catalytic subunit of the human pol gamma. A year later we were the first to clone part of the human po theta, the second family A eukaryotic DNA polymerase. Pol theta has been shown to conduct translesion DNA synthesis opposite an AP site or thymine glycol and has recently been implicated in base-excision repair (BER) of DNA damage. This year we demonstrated that Pol theta has intrinsic dRP lyase activity that is involved in single-nucleotide base excision DNA repair. Full-length human Pol &#952;is a 300-kDa polypeptide, but we demonstrated that the 98-kDa C-terminal region of Pol theta possesses both DNA polymerase activity and dRP lyase activity and is sufficient to carry out base excision repair in vitro. The 5'-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5'-dRP lyase activity. Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH4 cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol theta. These results are consistent with a role of Pol theta in BER. Since pol theta and gamma are both family A polymerases we propose that POLG dRP lyase active site must reside in a homologous site as that of pol theta.